The present application is the national stage under 35 U.S.C. xc2xa7371 of PCT/US96/10521, filed Jun. 14, 1996.
1. Field of the Invention
The present invention is generally in the field of receptors belonging to the TNF/NGF superfamily of receptors and the control of their biological functions. The TNF/NGF superfamily of receptors includes receptors such as the p55 and p75 tumor necrosis factor receptors (TNF-Rs, hereinafter called p55-R and p75-R) and the FAS ligand receptor (also called FAS/APO1 or FAS-R and hereinafter will be called FAS-R) and others. More specifically, the present invention concerns novel proteins which bind to the protein MORT-1 (or FADD), and more specifically, it relates to one such MORT-1 binding protein, herein designated MACH.
Accordingly, the present invention concerns, in general, new proteins which are capable of modulating or mediating the function of MORT-1 or of other proteins which bind to MORT-1 directly or indirectly. In particular, the present invention concerns MACH, its preparation and uses thereof,.as well as the various novel isoforms of MACH, their preparation and uses.
2. Background of the Related Art
Tumor Necrosis Factor (TNF-xcex1) and Lymphotoxin (TNF-xcex2) (hereinafter, TNF, refers to both TNF-xcex1 and TNF-xcex2) are multifunctional pro-inflammatory cytokines formed mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). Both TNF-xcex1 and TNF-xcex2 initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example, tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury. Thus, TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells. TNF can also be used as an anti-infectious agent.
However, both TNF-xcex1 and TNF-xcex2 also have deleterious effects. There is evidence that overproduction of TNF-xcex1 can play a major pathogenic role in several diseases. For example, effects of TNF-xcex1, primarily on the vasculature, are known to be a major cause for symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF-xcex1 was thus called cachetin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the process of inflammation and in many other diseases.
Two distinct, independently expressed, receptors, the p55 and p75 TNF-Rs, which bind both TNF-xcex1 and TNF-xcex2 specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, the cellular mechanisms, for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the p55 an p75 TNF-Rs have yet to be elucidated. It is this intracellular signaling, which occurs usually after the binding of the ligand, i.e., TNF (xcex1 or xcex2), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
As regards the above-mentioned cytocidal effect of TNF, in most cells studied so far, this effect is triggered mainly by the p55 TNF-R. Antibodies against the extracellular domain (ligand binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which correlates with the effectivity of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process. Further, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of the p55 TNF-R depends on the integrity of its intracellular domain, and accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of the p55 TNF-R. Moreover, TNF (xcex1 and xcex2) occurs as a homotrimer, and as such, has been suggested to induce intracellular signaling via the p55 TNF-R by way of its ability to bind to and to cross-link the receptor molecules, i.e., cause receptor aggregation.
Another member of the TNF/NGF superfamily of receptors is the FAS receptor (FAS-R) which has also been called the FAS antigen, a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R. The FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991), and appears to serve as a negative selector of autoreactive T cells, i.e., during maturation of T cells, FAS-R mediates the apoptopic death of T cells recognizing self-antigens. It has also been found that mutations in the FAS-R gene (lpr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for the FAS-R appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytesxe2x80x94CTLs), and hence when such CTLs contact cells carrying FAS-R, they are capable of inducing apoptopic cell death of the FAS-R-carrying cells. Further, a monoclonal antibody has been prepared that is specific for FAS-R, this monoclonal antibody being capable of inducing apoptopic cell death in cells carrying FAS-R, including mouse cells transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
While some of the cytotoxic effects of lymphocytes are mediated by interaction of a lymphocyte-produced ligand with the widely occurring cell surface receptor FAS-R (CD95), which has the ability to trigger cell death, it has also been found that various other normal cells, besides T lymphocytes, express the FAS-R on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, finding ways to restrain the cytotoxic activity of FAS-R may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry the FAS-R on their surface, antibodies against FAS-R, or the FAS-R ligand, may be used to trigger the FAS-R mediated cytotoxic effects in these cells and thereby provide a means for combating such malignant cells or HIV-infected cells (see Itoh et al., 1991). Finding yet other ways for enhancing the cytotoxic activity of FAS-R may therefore also have therapeutic potential.
It has been a long felt need to provide a way for modulating the cellular response to TNF (xcex1 or xcex2) and FAS-R ligand. For example, in the pathological situations mentioned above, where TNF or FAS-R ligand is overexpressed, it is desirable to inhibit the TNF- or FAS-R ligand-induced cytocidal effects, while in other situations, e.g., wound healing applications, it is desirable to enhance the TNF effect, or in the case of FAS-R, in tumor cells or HIV-infected cells, it is desirable to enhance the FAS-R mediated effect.
A number of approaches have been made by the laboratory of the applicants (see for example, European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (being essentially the soluble extracellular domains of the receptors) to compete with the binding of TNF to the cell surface-bound TNF-Rs. Further, on the basis that TNF-binding to its receptors is required for the TNF-induced cellular effects, approaches by the laboratory of of the applicants (see for example EPO 568925) have been made to modulate the TNF effect by modulating the activity of the TNF-Rs.
Briefly, EPO 568925 relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal function of the TNF-Rs. In EPO 568925, there is described the construction and characterization of various mutant p55 TNF-Rs, having mutations in the extracellular, transmembrane, and intracellular domains of the p55 TNF-R. In this way, regions within the above domains of the p55 TNF-R were identified as being essential to the functioning of the receptor, i.e., the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effect on the cells. Further, there is also described a number of approaches to isolate and identify proteins, peptides or other factors which are capable of binding to the various regions in the above domains of the TNF-R, which proteins, peptides and other factors may be involved in regulating or modulating the activity of the TNF-R. A number of approaches for isolating and cloning the DNA sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with the TNF-R or with the above proteins and peptides that bind various regions of the TNF-R, are also set forth in EPO 568925. However, EPO 568925 does not specify the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs (e.g., p55 TNF-R), nor does it describe the yeast two-hybrid approach to isolate and identify such proteins or peptides which bind to the intracellular domains of TNF-Rs. Similarly, heretofore there has been no disclosure of proteins or peptides capable of binding the intracellular domain of FAS-R.
Thus, when it is desired to inhibit the effect of TNF, or the FAS-R ligand, it would be desirable to decrease the amount or the activity of TNF-Rs or FAS-R at the cell surface, while an increase in the amount or the activity of TNF-Rs or FAS-R would be desired when an enhanced TNF or FAS-R ligand effect is sought. To this end the promoters of both the p55 TNF-R and the p75 TNF-R have been sequenced, analyzed and a number of key sequence motifs have been found that are specific to various transcription regulating factors, and as such the expression of these TNF-Rs can be controlled at their promoter level, i.e., inhibition of transcription from the promoters for a decrease in the number of receptors, and an enhancement of transcription from the promoters for an increase in the number of receptors (EP 606869 and WO 9531206). Corresponding studies concerning the control of FAS-R at the level of the promoter of the FAS-R gene have yet to be reported.
While it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor FAS-R, trigger in cells, upon stimulation by leukocyte-produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in FAS-R and the p55 TNF receptor (p55-R) signaling for cytotoxicity involve distinct regions within their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions (the xe2x80x98death domainsxe2x80x99) have sequence similarity. The xe2x80x98death domainsxe2x80x99 of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes that receptor aggregation which is necessary for initiation of signaling (see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), and at high levels of receptor expression can result in triggering of ligand-independent signaling (Bolding et al., 1995).
Thus, prior to WO 9531544 and the present invention, there have not been provided proteins which may regulate the effect of ligands belonging to the TNF/NGF superfamily, such as the TNF or FAS-R ligand effect on cells, by mediation of the intracellular signaling process, which signaling is believed to be governed to a large extent by the intracellular domains (ICs) of the receptors belonging to the TNF/NGF superfamily of receptors, such as those of the TNF-Rs, i.e. the p55 and p75 TNF-R intracellular domains (p55IC and p75IC, respectively), as well as the FAS-IC.
Some of the cytotoxic effects of lymphocytes are mediated by interaction of a lymphocyte-produced ligand with FAS-R (CD-95), a widely occurring cell surface receptor which has the ability to trigger cell death (see Nagata and Golstein, 1995). Cell killing by mononuclear phagocytes involves a ligand-receptor couple, TNF and its receptor p55-R (CD120), that is structurally related to FAS-R and its ligand (see also Vandenabeele et al., 1995). Like other receptor-induced effects, cell death induction by the TNF receptors and FAS-R occurs via a series of protein-protein interactions, leading from ligand-receptor binding to the eventual activation of enzymatic effector functions, which in the case of these particular receptors results in cell death. Previous studies have elucidated non-enzymatic protein-protein interactions that initiate signaing for cell death: binding of trimeric TNF or the FAS-R ligand molecules to the receptors, the resulting interactions of their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993) augmented by a propensity of the death-domain motifs to self-associate, (Boldin et al., 1995a), and induced binding of two cytoplasmic proteins (which can also bind to each other) to the receptors"" intracellular domainsxe2x80x94MORT-1 (or FADD) to FAS-R (Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD to p55-R (Hsu et al., 1995; Hsu et al., 1996).
Three proteins that bind to the intracellular domain of FAS-R and p55-R at the xe2x80x9cdeath domainxe2x80x9d region involved in cell-death induction by the receptors through hetero-association of homologous regions and that independently are also capable of triggering cell death were identified by the yeast two-hybrid screening procedure. One of these is the protein, MORT-1 (Boldin et al. 1995b) also known as FADD (Chinnaiyan et al., 1995), that binds specifically to FAS-R. A second one, TRADD (see also Hsu et al., 1995, 1996), binds to p55-R, and the third, RIP (see also Stanger et al., 1995), binds to both FAS-R and p55-R. Besides their binding to FAS-R and p55-R, these proteins are also capable of binding to each other, which provides for a functional xe2x80x9ccross-talkxe2x80x9d between FAS-R and p55-R. These bindings occur through a conserved sequence motif, the xe2x80x9cdeath domain modulexe2x80x9d common to the receptors and their associated proteins. Furthermore, although in the yeast two-hybrid test MORT-1 was shown to bind spontaneously to FAS-R, in mammalian cells this binding takes place only after stimulation of the receptor, suggesting that MORT-1 participates in the initiating events of FAS-R signaling. MORT-1 does not contain any sequence motif characteristic of enzymatic activity, and therefore, its ability to trigger cell death seems not to involve an intrinsic activity of MORT-1 itself, but rather, activation of some other protein(s) that bind MORT-1 and act further downstream in the signaling cascade. Cellular expression of MORT-1 mutants lacking the N-terminal part of the molecule has been shown to block cytotoxicity induction by FAS/APO1 (FAS-R) or p55-R (Hsu et al., 1996; Chinnaiyan et al., 1996), indicating that this N-terminal region transmits the signaling for the cytocidal effect of both receptors through protein-protein interactions.
Recent studies have implicated a group of cytoplasmic thiol proteases which are structurally related to the Caenorhabditis elegans protease CED3 and to the mammalian interleukin-1xcex2 converting enzyme (ICE) in the onset of various physiological cell death processes (reviewed in Kumar, 1995 and Henkart, 1996). There have also been some indications that protease(s) of this family may take part in the cell-cytotoxicity induced by FAS-R and TNF-Rs. Specific peptide inhibitors of the proteases and two virus-encoded proteins that block their function, the cowpox protein crmA and the Baculovirus p35 protein, were found to provide protection to cells against this cell-cytotoxicity (Enari et al., 1995; Los etal., 1995; Tewari et al., 1995; Xue et al., 1995; Beidler et al., 1995). Rapid cleavage of certain specific cellular proteins, apparently mediated by protease(s) of the CED3/ICE family, was observed in cells shortly after stimulation of FAS-R or TNF-Rs. Heretofore, no information has been presented as to the identity of the specific CED3/ICE-related protease(s) involved, nor of the mechanisms of activation of these protease(s) by the receptors.
It is an object of the invention to provide novel proteins, including all isoforms, analogs, fragments or derivatives thereof, which are capable of binding to MORT-1, which itself binds to the intracellular domain of the FAS-R, which novel proteins affect the intracellular signaling process initiated by the binding of FAS ligand to its receptor.
Another object of the invention is to provide antagonists (e.g., antibodies, peptides, organic compounds, or even some isoforms) to the above novel proteins, analogs, fragments and derivatives thereof, which may be used to inhibit the signaling process, or, more specifically, the cell-cytotoxicity, when desired.
A further object of the invention is to use the above novel proteins, analogs, fragments and derivatives thereof, to isolate and characterize additional proteins or factors, which may be involved in regulation of receptor activity, e.g., other proteases which cleave the novel proteins to render them biologically active, and/or to isolate and identify other receptors further upstream in the signaling process to which these novel proteins, analogs, fragments and derivatives bind (e.g., other FAS-Rs or related receptors), and hence, in whose function they are also involved.
A still further object of the invention is to provide inhibitors which can be introduced into cells to bind or interact with the MACH proteases and inhibit their proteolytic activity.
Moreover, it is an object of the present invention to use the above-mentioned novel proteins, and analogs, fragments and derivatives thereof as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies, in turn, may be used, for example, for the purification of the new proteins from different sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g., for identifying disorders related to abnormal functioning of cellular effects mediated by the FAS-R or other related receptors.
A further object of the invention is to provide pharmaceutical compositions comprising the above novel proteins, or analogs, fragments or derivatives thereof, as well as pharmaceutical compositions comprising the above noted antibodies or other antagonists.
In accordance with the present invention, a novel protein, MACH, which is capable of binding to, or interacting with, MORT-1, which itself binds to the intracellular domain of the FAS-R was discovered. MACH probably functions as an effector component of the cell-death pathway initiated by the binding of FAS ligand to FAS-R at the cell surface, and this by virtue of the fact that at least some of the isoforms of MACH appear to be active intracellular proteases. Proteases of the CED3/ICE family have been implicated in the apoptopic process triggered by FAS-R. MORT-1 (or FADD) binds to the intracellular domain of FAS-R upon activation of this receptor and the novel MACH proteins of the present invention bind to MORT-1. The MACH protein, cloned and characterized in accordance with the present invention, actually exists in multiple isoforms, some of which isoforms have a CED3/ICE homology region which has proteolytic activity (proteolytic domain), and causes the death of cells when expressed in the cells. Thus, activation of this novel CED3/ICE homolog (i.e., the various MACH isoforms having the proteolytic domain) by FAS-R (via MORT-1 interaction) appears to constitute an effector component of the FAS-R-mediated cell-death pathway.
Moreover, MACH also appears to function as an effector component of the cell-death pathway initiated by the binding of TNF to p55-R at the cell surface, this by way of indirect mechanism of MORT-1 binding to TRADD, a protein which binds to the intracellular domain of p55-R (Hsu et al., 1995), followed by or together with MACH binding to MORT-1, with the activation of MACH into an active protease involved in effecting cell death.
It should also be noted that while MACH, in particular, the MACHal isoform, displays all of the sequence features critical of the function of the CED3/ICE proteases, it does, however, have some distinctive sequence features of its own which may endow it with a unique and possibly tissue/cell specific mode of action.
MORT-1 (for xe2x80x98Mediator of Receptor Toxicityxe2x80x99, Boldin et al., 1995b), previously designated HF1, is capable of binding to the intracellular domain of the FAS-R. This FAS-IC-binding protein appear to act as a mediator or modulator of the FAS-R ligand effect on cells by way of mediating or modulating the intracellular signaling process which usually occurs following the binding of the FAS-R ligand at the cell surface. In addition to its FAS-IC-binding specificity, MORT-1 was shown to have other characteristics (see Example 1), for example, it has a region homologous to the xe2x80x9cdeath domainxe2x80x9d (DD) regions of the p55-TNF-R and FAS-R (p55-DD and FAS-DD), and thereby is also capable of self-association. MORT-1 is also capable of activating cell cytotoxicity on its own, an activity possibly related to its self-association capability. It has now also been found that co-expression of the region in MORT-1 (HF1) that contains the xe2x80x9cdeath domainxe2x80x9d homology sequence (MORT-DD, present in the C-terminal part of MORT-1) strongly interferes with FAS-induced cell death, as would be expected from its ability to bind to the xe2x80x9cdeath domainxe2x80x9d of the FAS-IC. Further, in the same experimental conditions, it was found that co-expression of the part of MORT-1 that does not contain the MORT-DD region (the N-terminal part of MORT-1, amino acids 1-117, xe2x80x9cMORT-1 headxe2x80x9d) resulted in no interference of the FAS-induced cell death and, if at all, a somewhat enhanced FAS-induced cell cytotoxicity.
Accordingly, it is likely that MORT-1 also binds to other proteins involved in the intracellular signaling process. These MORT-1-binding proteins may therefore also act as indirect mediators or modulators of the FAS-R ligand effect on cells by way of mediating or modulating the activity of MORT-1; or these MORT-1-binding proteins may act directly as mediators or modulators of the MORT-1-associated intracellular signaling process by way of mediating or modulating the activity of MORT-1, which, as noted above, has an apparently independent ability to activate cell cytotoxicity. These MORT-1-binding proteins may also be used in any of the standard screening procedures to isolate, identify and characterize additional proteins, peptides, factors, antibodies, etc., which may be involved in the MORT-1-associated or FAS-R-associated signaling process or may be elements of other intracellular signaling processes. Such MORT-1-binding proteins have been isolated and are described herein (see Example 2 and Example 3). One of these MORT-1-binding proteins, herein designated MACH, was initially cloned, sequenced, and partially characterized as having the following properties: The MACH cDNA encodes the ORF-B open-reading frame; MACH binds to MORT-1 in a very strong and specific manner; the MACH binding site in MORT-1 occurs upstream of the MORT-1 xe2x80x9cdeath domainxe2x80x9d motif; the ORF-B region of MACH is the MORT-1-interacting part thereof; and MACH is capable of self-association and of inducing cell-cytotoxicity on its own.
In accordance with the present invention, it has now been shown as mentioned above, that MACH actually exists in a number of isoforms. Moreover, the MACH ORF-B noted above is in fact one of the MACH isoforms designated herein as MACHxcex21 (see below).
Accordingly, the present invention provides a DNA sequence encoding a protein, analogs or fragments thereof, capable of binding to or interacting with MORT-1, said protein, analogs or fragments thereof being capable of mediating the intracellular effect mediated by the FAS-R or p55-TNF-R.
In particular, the present invention provides a DNA sequence selected from the group consisting of:
(a) a cDNA sequence derived from the coding region of a native MORT-1 binding protein;
(b) DNA sequences capable of hybridization to a sequence of (a) under moderately stringent conditions and which encode a biologically active MORT-1 binding protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active MORT-1 binding protein.
Another specific embodiment of the above DNA sequence of the invention is a DNA sequence comprising at least part of the sequence encoding at least one isoform of the MACH protein selected from the herein designated MACH isoforms MACHxcex11, MACHxcex12, MACHxcex13, MACHxcex22, MACHxcex21, MACHxcex23, MACHxcex24 and MACHxcex25.
Other specific embodiments of the DNA sequence of the invention as noted above are DNA sequences encoding:
(a) a MACH isoform selected from MACHxcex11, MACHxcex21 and MACHxcex23 having an amino acid sequence set forth in SEQ ID NOs:7, 5 and 8 respectively, and analogs and fragments of any one thereof;
(b) MACHxcex11 having the amino acid sequence set forth in SEQ ID NO:7, and analogs and fragments thereof;
(c) MACHxcex21 having the amino acid sequence set forth in SEQ ID NO:5, and analogs and fragments thereof;
(d) MACHxcex23 having the amino acid sequence set forth in SEQ ID NO:8, and analogs and fragments thereof.
In the present invention provides MORT-1-binding proteins, and analogs, fragments or derivatives thereof encoded by any of the above sequences of the invention, said proteins, analogs, fragments and derivatives being capable of binding to or interacting with MORT-1 and mediating the intracellular effect mediated by the FAS-R or p55 TNF-R.
A specific embodiment of the invention is the MORT-1-binding protein, analogs fragments and derivatives thereof, which are selected from as least one isoform of MACH of the group comprising MACHxcex11, MACHxcex12, MACHxcex13, MACHxcex21, MACHxcex22, MACHxcex23, MACHxcex24 and MACHxcex25 which have at least part of the amino acid sequences thereof.
Also provided by the present invention are vectors encoding the above MORT-1-binding protein, and analogs, fragments or derivatives of the invention, which contain the above DNA sequence of the invention, these vectors being capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells containing such vectors; and a method for producing the MORT-1-binding protein, or analogs, fragments or derivatives of the invention by growing such transformed host cells under conditions suitable for the expression of said protein, analogs, fragments or derivatives, effecting post-translational modifications of said protein as necessary for obtaining said protein and extracting said expressed protein, analogs, fragments or derivatives from the culture medium of said transformed cells or from cell extracts of said transformed cells. The above definitions are intended to include all isoforms of the MACH protein.
In another aspect, the present invention also provides antibodies or active derivatives or fragments thereof specific the MORT-1-binding protein, and analogs, fragments and derivatives thereof, of the invention.
By yet another aspect of the invention, there are provided various uses of the above DNA sequences or the proteins which they encode, according to the invention, which uses include amongst others:
(i) A method for the modulation of the FAS-R ligand or TNF effect on cells carrying a FAS-R or p55-R, comprising treating said cells with one or more MORT-1-binding proteins, analogs, fragments or derivatives of the invention, capable of binding to MORT-1, which binds to the intracellular domain of FAS-R, or capable of binding to MORT-1 which binds to TRADD which binds to the intracellular domain of p55-R, and thereby being capable of modulating/mediating the activity of said FAS-R or p55 TNF-R, wherein said treating of said cells comprises introducing into said cells said one or more proteins, analogs, fragments or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(ii) A method for the modulation of the FAS-R ligand or TNF effect on cells according to (i) above, wherein said treating of cells comprises introducing into said cells said MORT-1-binding protein, or analogs, fragments or derivatives thereof, in a form suitable for intracellular introduction, or introducing into said cells a DNA sequence encoding said MORT-1-binding protein, or analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(iii) A method as in (ii) above wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of a FAS-R- or p55-R-carrying cell and a second sequence encoding a protein selected from MORT-1-binding protein, and analogs, fragments and derivatives thereof, that when expressed in said cells is capable of modulating/mediating the activity of said FAS-R or p55-R; and
(b) infecting said cells with said vector of (a).
(iv) A method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or a p55-R comprising treating said cells with antibodies or active fragments or derivatives thereof, according to the invention, said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells, wherein when the MORT-1-binding protein, or portions thereof of said cells are exposed on the extracellular surface, said composition is formulated for extracellular application, and when said MORT-1-binding proteins are intracellular, said composition is formulated for intracellular application.
(v) A method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or p55-R comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence of at least part of the MORT-1-binding protein sequence of the invention, said oligonucleotide sequence being capable of blocking the expression of the MORT-1-binding protein.
(vi) A method as in (ii) above for treating tumor cells or HIV-infected cells or other diseased cells, comprising:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a protein selected from MORT-1-binding protein, analogs, fragments and derivatives of the invention, that when expressed in said tumor, HIV-infected, or other diseased cell is capable of killing said cell; and
(b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).
(vii) A method for modulating the FAS-R ligand or TNF effect on cells comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a MORT-1-binding protein according to the invention, is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said MORT-1-binding protein in said cells.
(viii) A method selected from the method according to the invention, wherein said MORT-1-binding protein encoding sequence comprises at least one of the MACH isoforms, analogs, fragments and derivatives of any thereof according to the invention which are capable of binding specifically to MORT-1 which in turn binds specifically to FAS-IC, or which are capable of binding to MORT-1 which in turn binds to TRADD and which in turn binds to the p55-IC.
(ix) A method for isolating and identifying proteins, according to the invention, capable of binding to the MORT-1 protein, comprising applying the yeast two-hybrid procedure in which a sequence encoding said MORT-1 protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said MORT-1 protein, said protein being the MORT-1-binding proteins.
(x) A method according to any one of (i)-(ix) above wherein said MORT-1-binding protein is the MACH isoform herein designated MACHxcex11, analogs, fragments and derivatives thereof.
(xi) A method according to any one of (i)-(ix) above wherein said MORT-1-binding protein is the MACH isoform herein designated MACHxcex21, analogs, fragments and derivatives thereof.
(xii) A method according to any one of (i)-(ix) above wherein said MORT-1-binding protein is the MACH isoform herein designated MACHxcex23, analogs, fragments and derivatives thereof.
The present invention also provides a pharmaceutical composition for the modulation of the FAS-R ligand- or TNF-effect on cells comprising, as active ingredient any one of the following:
(i) a MORT-1-binding protein according to the invention, and biologically active fragments, analogs, derivatives or mixtures thereof;
(ii) a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding a MORT-1-binding protein or biologically active fragments or analogs, according to the invention;
(iii) an oligonucleotide sequence encoding an anti-sense sequence of the MORT-1-binding protein sequence according to the invention, wherein said oligonucleotide may be the second sequence of the recombinant animal virus vector of (ii) above.
The present invention also provides:
I. a method for the modulation of the MORT-1-induced effect or MORT-1-binding protein-induced effect on cells comprising treating said cells in accordance with a method of any one of (i)-(xi) above, with MORT-1-binding proteins, analogs, fragments or derivatives thereof or with sequences encoding MORT-1-binding proteins, analogs or fragments thereof, said treatment resulting in the enhancement or inhibition of said MORT-1-mediated effect, and thereby also of the FAS-R or p55-R-mediated effect.
II. a method as above wherein said MORT-1-binding protein, analog, fragment or derivative thereof is that part of the MORT-1-binding protein which is specifically involved in binding to MORT-1 or MORT-1-binding protein itself, or said MORT-1-binding protein sequence encodes that part of MORT-1-binding protein which is specifically involved in binding to MORT-1 or the MORT-1-binding protein itself.
III. A method as above wherein said MORT-1-binding protein is any one of the MACH isoforms selected from MACHxcex11, MACHxcex21, and MACHxcex23, said MACH isoforms capable of enhancing the MORT-1-associated effect on cells and thereby also of enhancing the FAS-R- or p55-R-associated effect on cells.
As arises from all the above-mentioned, as well as from the detailed description hereinbelow, MACH may be used in a MORT-1-independent fashion to treat cells or tissues. Isolation of the MORT-1-binding proteins, their identification and characterization may be carried out by any of the standard screening techniques used for isolating and identifying proteins, for example, the yeast two-hybrid method, affinity chromatography methods, and any of the other well-known standard procedures used for this purpose.
Other aspects and embodiments of the present invention are also provided as arising from the following detailed description of the invention.
It should be noted that, where used throughout, the following terms: xe2x80x9cModulation of the FAS-ligand or TNF effect on cellsxe2x80x9d; and xe2x80x9cModulation of the MORT-1 or MORT-1-binding protein effect on cellsxe2x80x9d are understood to encompass in vitro as well as in vivo treatment.